Abstract

AIM:

Peroxisome proliferator activated receptor gamma (PPARgamma) agonists have been shown to prevent hepatic fibrosis in rodents. We evaluated the therapeutic antifibrotic potential of the PPARgamma agonist pioglitazone on established hepatic fibrosis.

METHODS:

Repeated injections of carbon tetrachloride (CCl4), a choline deficient diet, or bile duct ligation (BDL) were used to induce hepatic fibrosis in rats. Pioglitazone treatment was introduced at various time points. Therapeutic efficacy was assessed by comparison of the severity of hepatic fibrosis in pioglitazone treated versus untreated fibrotic controls.

RESULTS:

When introduced after two weeks of CCl4, pioglitazone reduced hepatic fibrosis, OH proline content, hepatic mRNA expression of collagen type I, and profibrotic genes, as well as the number of activated alpha smooth muscle actin positive hepatic stellate cells, compared with rats receiving CCl4 only, with no significant change in necroinflammation. When pioglitazone treatment was initiated after five weeks of CCl4, no antifibrotic effect was observed. Similarly, pioglitazone was associated with a reduced severity of fibrosis induced by a choline deficient diet when introduced early, while delayed treatment with pioglitazone remained ineffective. In contrast, pioglitazone failed to interrupt progression of fibrosis due to BDL, irrespective of the timing of its administration.

CONCLUSION:

In rats, the therapeutic antifibrotic efficacy of pioglitazone is limited and dependent on the type of injury, duration of disease, and/or the severity of fibrosis at the time of initiation of treatment.

Experimental design for evaluation of the therapeutic potential of pioglitazone (PGZ) on established hepatic fibrosis. Hepatic fibrosis induced by (A) repeated intraperitoneal injections of carbon tetrachloride (CCl4 0.75 ml/kg, twice weekly), (B) chronic administration of a choline deficient L‐amino acid (CDAA) defined diet, or (C) bile duct ligation (BDL). PGZ treatment was initiated at the indicated times and delivered as a food mixture (0.01% wt/wt). In experiments (A) and (B), five rats per group were analysed and in (C) there were four rats in each group.

Pioglitazone (PGZ) is biologically active in carbon tetrachloride (CCl4) injected rats. Expression of CD36 mRNA in adipose tissue in rats treated with CCl4 only, and in rats treated with PGZ for the last three weeks of the five week CCl4 regimen or the last four weeks of the nine week CCl4 regimen, determined by real time polymerase chain reaction. Control rats were fed the PGZ supplemented diet for two weeks as a positive control for PGZ effects. Values are normalised to expression of RLP19 mRNA, regarded as an internal control, and expressed in relation to the mean value in untreated controls arbitrarily set at 1. Data are mean (SD) (n = 5/group). ***p<0.001 for groups treated with PGZ compared with groups not treated with PGZ at the same time point.

Effects of pioglitazone (PGZ) treatment on progression of carbon tetrachloride (CCl4) induced hepatic fibrosis. Representative photomicrographs of Sirius red stained liver sections from rats who received CCl4 for two weeks (A), for five weeks without (B) or with (C) PGZ treatment for the last three weeks, for nine weeks without (D) or together with (E) PGZ treatment for the last four weeks (E). Original magnification 5×. Quantification of hepatic hydroxy (OH) proline content (F) in rats injected twice weekly with CCl4 for the indicated periods of time and in rats treated with PGZ for the last three weeks of the five week CCl4 regimen or the last four weeks of the nine week CCl4 regimen (CCl4 + PGZ). Data are mean (SD) for n = 5/group.***p<0.001 compared with 2 wk CCl4 livers; †p<0.05, ††p<0.01 compared with similar treatment at five weeks.

Effects of pioglitazone (PGZ) treatment on the distribution and quantification of α smooth muscle actin (α‐SMA) positive hepatic cells during progression of carbon tetrachloride (CCl4) induced hepatic fibrosis. Representative photomicrographs of α‐SMA immunostained liver sections from rats receiving CCl4 for two weeks (A), for five weeks without (B) or with (C) PGZ treatment for the last three weeks, for nine weeks without (D) or together with (E) PGZ treatment for the last four weeks. P, portal tract, C, central vein. Note that no α‐SMA positive cells were found in the thick fibrotic bundles (arrows). Original magnification 5×. (F) Quantification of hepatic area occupied by α‐SMA positive cells in rats injected twice weekly with CCl4 for the indicated periods of time and in rats treated with PGZ for the last three weeks of the five week CCl4 regimen or the last four weeks of the nine week CCl4 regimen (CCl4 + PGZ). Data are expressed as mean (SD) percentage of α‐SMA positive area related to the total area of the section for n = 5/group. **p<0.01, ***p<0.001 compared with 2 wk CCl4 livers.

Effects of pioglitazone (PGZ) on hepatic collagen type I (Col), tissue inhibitor of metalloproteinase (TIMP‐1), Kruppel‐like factor 6 (KLF‐6), transforming growth factor β1 (TGF‐β1), and connective tissue growth factor (CTGF) mRNA levels during progression of carbon tetrachloride (CCl4) induced hepatic fibrosis. Expression of type I collagen (A), T IMP‐1 (B), KLF‐6 (C), TGF‐β1 (D), and CTGF (E) mRNA determined by real time polymerase chain reaction in the liver of rats treated with CCl4 alone or together with PGZ for the last three weeks of the five week CCl4 regimen or the last four weeks of the nine week CCl4 regimen. Values are normalised to expression of RLP19 mRNA, regarded as an invariant control, and expressed in relation to the mean value in untreated controls arbitrarily set at 1. Data are mean (SD) (n = 5/group). There were no significant differences between values obtained in 9 wk CCl4 rats, treated or not with PGZ.

Effect of pioglitazone (PGZ) on the cleaved and glycoforms of matrix metalloproteinase 13 (MMP‐13) in the liver of carbon tetrachloride (CCl4) rats. Mean optical density of the glycosylated inactive form of MMP‐13 and the cleaved form of MMP‐13. Separation by electrophoresis of total liver homogenate in non‐denaturing conditions resolved the glycosylated form at 65–70 kDa and the cleaved MMP‐13 at 45–50 kDa (see methods). Data are mean for n = 5 per group, in duplicate, with 100% arbitrarily attributed to the sum of the glycosylated and cleaved forms of MMP‐13 in control livers. ***p<0.001 compared with control livers; †p<0.05 compared with five week CCl4 livers under similar conditions. There were no significant differences between values obtained in 9 wk CCl4 rats, treated or not with PGZ.

Effects of pioglitazone (PGZ) on serum levels of adiponectin in carbon tetrachloride (CCl4) treated rats. Serum adiponectin was determined by radioimmunoassay and is presented as mean (SD) for n = 5 per group in rats injected twice weekly with CCl4 for the indicated periods of time and in rats treated with PGZ for the last three weeks of the five week CCl4 regimen or the last four weeks of the nine week CCl4 regimen. Control rats were fed the PGZ supplemented diet for two weeks as a positive control for PGZ effect. ***p<0.001 for groups treated with PGZ compared with groups not treated with PGZ at the same times of CCl4 injections. †p<0.05 in 9 wk CCl4 rats compared with other times of CCl4 injection.

Effects of a choline deficient L‐amino acid (CDAA) diet and pioglitazone (PGZ) on total liver lipid content. Total lipid content in the liver of rats fed CDAA for the indicated time periods or receiving the PGZ supplemented CDAA for the last four weeks of the six week regimen or for the last six weeks of the 12 week regimen. Data are mean (SD) for n = 5 per group. **p<0.01, ***p<0.001 compared with 0 wk CDAA (controls); †p<0.05, ††p<0.01 compared with 2 wk CDAA fed rats.

Effects of pioglitazone (PGZ) treatment on collagen deposit, activation of hepatic stellate cells, and expression of collagen I, connective tissue growth factor (CTGF), and transforming growth factor β1 (TGF‐β1) in choline deficient L‐amino acid (CDAA) induced hepatic fibrosis. Quantification of hepatic hydroxy (OH) proline content (A), quantification of hepatic area occupied by α smooth muscle actin (α‐SMA) positive cells expressed as percentage of the total hepatic section (B), and hepatic type I collagen (C), CTGF (D), and TGF‐β1 (E) mRNA determined by real time polymerase chain reaction in rats fed the CDAA diet for the indicated times or who received the PGZ supplemented CDAA diet for the last four weeks of the six week regimen or for the last six weeks of the 12 week regimen. Values were normalised to expression of RLP19 mRNA regarded as an internal control, and expressed in relation to the mean value in untreated controls arbitrarily set at 1. All data are expressed as mean (SD) for n = 5/group. *p<0.05, **p<0.01, ***p<0.001 compared with 0 wk CDAA (controls).

Effects of pioglitazone (PGZ) on serum level of adiponectin and adipose expression of CD36 mRNA in choline deficient L‐amino acid (CDAA) fed rats. (A) Serum adiponectin determined by radioimmunoassay. (B) CD36 mRNA in adipose tissue determined by real time polymerase chain reaction in rats fed the CDAA diet for the indicated time periods (n = 5 per group) or who received the PGZ supplemented CDAA diet for the last four weeks of the six week regimen or for the last six weeks of the 12 week regimen (n = 6 per group). Control rats were fed the PGZ supplemented diet for two weeks as a positive control for PGZ effect. Data are mean (SD).

Effects of pioglitazone (PGZ) treatment on the distribution and quantification of α smooth muscle actin (α‐SMA) positive hepatic cells during progression of hepatic fibrosis induced by bile duct ligation (BDL). Representative photomicrographs of α‐SMA immunostained liver sections from rats two weeks (A) or four weeks (B) after BDL, or from rats who received a PGZ supplemented diet from day 5 to day 14 post‐BDL (BDL 2 wk + PGZ) (C) or from day 14 to day 28 post BDL (BDL 4 wk + PGZ) (D). Original magnification 10×. Quantification of hepatic area occupied by α‐SMA positive cells in the different treatment groups (E). Data are expressed as the mean (SD) percentage of α‐SMA positive area related to the total area of the section for n = 4/group. **p<0.01 compared with 2 wk BDL livers similarly treated.